Therapeutic beta-lactam dosages and broad-spectrum antibiotics are associated with reductions in microbial richness and diversity in persons with cystic fibrosis

Persons with cystic fibrosis (PwCF) suffer from pulmonary exacerbations (PEx) related in part to lung infection. While higher microbial diversity is associated with higher lung function, the data on the impact of short-term antibiotics on changes in microbial diversity is conflicting. Further, Prevotella secretes beta-lactamases, which may influence recovery of lung function. We hypothesize that sub-therapeutic and broad spectrum antibiotic exposure leads to decreasing microbial diversity. Our secondary aim was to evaluate the concerted association of beta-lactam pharmacokinetics (PK), antibiotic spectrum, microbial diversity, and antibiotic resistance on lung function recovery using a pathway analysis. This was a retrospective observational study of persons with CF treated with IV antibiotics for PEx between 2016 and 2020 at Children’s National Hospital; respiratory samples and clinical information were collected at hospital admission for PEx (E), end of antibiotic treatment (T), and follow-up (F). Metagenomic sequencing was performed; PathoScope 2.0 and AmrPlusPlus were used for taxonomic assignment of sequences to bacteria and antibiotic resistance genes (ARGs). M/W Pharm was used for PK modeling. Comparison of categorical and continuous variables and pathway analysis were performed in STATA. Twenty-two PwCF experienced 43 PEx. The study cohort had a mean age of 14.6 years. Only 12/43 beta-lactam courses had therapeutic PK, and 18/43 were broad spectrum. A larger decrease in richness between E and T was seen in the therapeutic PK group (sufficient − 20.1 vs. insufficient − 1.59, p = 0.025) and those receiving broad spectrum antibiotics (broad − 14.5 vs. narrow − 2.8, p = 0.030). We did not detect differences in the increase in percent predicted forced expiratory volume in one second (ppFEV1) at end of treatment compared to PEx based on beta-lactam PK (sufficient 13.6% vs. insufficient 15.1%) or antibiotic spectrum (broad 11.5% vs. narrow 16.6%). While both therapeutic beta-lactam PK and broad-spectrum antibiotics decreased richness between PEx and the end of treatment, we did not detect longstanding changes in alpha diversity or an association with superior recovery of lung function compared with subtherapeutic PK and narrow spectrum antimicrobials.

proteinase K) opening the bacterial cell walls/outer membranes to extract the bacterial DNA. Qubit (Thermofisher Scientific) and Bioanalyzer (Agilent) were used to measure DNA quantity and quality, respectively. DNA libraries for next generating sequencing (NGS) were constructed using a Nextera XT Library Prep Kit (Illumina). Between 23-30 libraries per run were sequenced on a NextSeq 500 (Illumina) using a Mid-Output 2x150 cycle kit. We had an average of 5.8 million reads/sample (range 670K to 21M). After filtering out the remaining human reads in the samples using KneadData [7], we were left with an average of 1.6 million reads/sample (range 38K to 8M). For each individual sample, our Goods coverage was 1, supporting that our sequencing depth was sufficient for subsequent analysis of microbial diversity measures.
Determination of beta-lactam antibiotic resistance genes. AMR Plus Plus [8] was used to identify betalactam resistance genes. This program uses the MEGARes database (https://megares.meglab.org/), which contains the sequence data for more than 8,000 antimicrobial resistance genes. Specifically for beta-lactams, this includes Class A-D beta-lactamases, mutant porin proteins, penicillin binding protein, and penicillin binding protein regulator. The resulting count table for beta-lactam resistance as an antibiotic class was used for subsequent statistical analyses.
Statistical analyses. The exogenous variables in the structural equation model were beta-lactam PK sufficient (yes/no) and antibiotic spectrum (broad versus narrow). The first endogenous variable in the model was alpha diversity of the respiratory sample obtained at PEx onset (E), and the model was run using species observed, the Shannon index, and the inverse Simpson index. The other two endogenous variables were the relative abundance of Prevotella sp. and the relative abundance of beta-lactam antibiotic resistance genes in the PEx sample (E). The outcome variables were the increase in the percent predicted forced expiratory volume in one second (ppFEV1) from PEx onset (E) to end of antibiotic treatment (T) and the percent recovery of ppFEV1 at the end of antibiotic treatment (T) compared to the best ppFEV1 in the 6 months prior to PEx onset. A maximum likelihood with missing values model was used, with default standard errors.

Supplemental Results
Beta-lactam PK determinations. In two instances, meropenem and ceftazidime were administered concurrently and in one instance, meropenem and piperacillin/tazobactam were administered concurrently, all for treatment of Burkholderia sp. In all situations, meropenem was considered as the primary beta-lactam as the organism was resistant to the other beta-lactams (n=2) or meropenem had lower MIC (n=1). In one instance, meropenem was discontinued after 9 days of treatment due to development of rash and the study participant was started on ceftazidime. As the organism was resistant to ceftazidime but susceptible to meropenem, meropenem was used as primary beta-lactam for analysis. Additionally, the antimicrobial susceptibility results from the Gram-negative organisms used to determine T>MIC were obtained from the concurrent PEx respiratory culture for 67% (n=29) of the PEx event. The other 33% (n=14) were based on a Gram-negative organism in a prior culture (P. aeruginosa, n=13 and S. maltophilia, n=1). Twelve beta-lactam treatment courses achieved a T>MIC associated with bacterial killing and thus considered PK sufficient, while the remaining 31 courses were considered PK insufficient. As S. aureus was also detected in 14 PEx cultures, of which 8 were MRSA, the combination of antibiotics administered was also assessed for therapy that also targeted S. aureus. The antibiotics selected did not cover for MRSA in one instance, but this course was already deemed PK insufficient and so no changes were required to our assigned groups.
Beta-lactam antibiotic resistance genes. More than 900 antibiotic resistance genes were identified in our sample cohort (please see the supplemental Excel spreadsheet). Of these, 292 (32% of the total genes detected) were directed against beta-lactams. 26,638 total sequence hits (1.9%) were for Class A beta-lactamases, which included BLA, BLAZ, CARB, CFX, CTX, GES, HERA, MAL, OXY, ROB, SCO, SFO, SHV, and TEM. Importantly, 6229 of those sequence hits were against CFX, with CfxA being the most notable beta-lactamase associated with Prevotella species [11][12][13]. 15,576 total hits (1.1%) were for penicillin binding proteins, which included MECA, MECC, MECI, PBPB1A, PBP1B, BPB2, PBPB2B, PBP2X, and PBP4B. Only 5576 (0.4%) total hits were for Class B (16), Class C (2982), or Class D (2577) betalactamases. 2402 hits (0.17%) were for mutant porin proteins. As the total number of sequence hits for antibiotic resistance genes was much lower than for bacterial species, we observed that the rarefaction curves for antibiotic class suggested good coverage for comparison across samples, whereas we did not have enough sequencing depth to look at the antibiotic genes specifically. As such, beta-lactam resistance as an antibiotic class was used for subsequent analyses.
Association between azithromycin or inhaled antibiotic use and culture results. Azithromycin and inhaled antibiotics including tobramycin and aztreonam are typically started as chronic therapies in persons with CF persistently infected with P. aeruginosa. Other studies have suggested these therapies can be associated with the growth of other pathogens. Using chi-square, we tested the association between these agents and the top 5 pathogens identified in clinical culture: MSSA, MRSA, P. aeruginosa, B. gladioli, and B. cepacia complex (Supplemental Table 7). In our study cohort, only P. aeruginosa was associated with the use of these chronic antibiotic therapies.

PEx Characteristics N=43 Concurrent respiratory virus results (n, %)
Not tested 21 (49%) Human rhino/enterovirus 4 (9%) Respiratory syncytial virus 2 (5%) Non-SARS coronavirus 2 (5%) Influenza A* 1 (2%) Influenza B 1 (2%) Parainfluenza* 1 (2%) *These two viruses were a co-infection in one study participant PEx, pulmonary exacerbation; CFTR, cystic fibrosis transmembrane conductance regulator; SARS, severe acute respiratory syndrome. . PK_sufficient, beta-lactam pharmacokinetic sufficient; BSvsNS, antibiotic spectrum; e_sobs, species observed at pulmonary exacerbation; Prevotella_E, relative abundance of Prevotella at pulmonary exacerbation; BL_ARG_E, relative abundance of beta-lactam antibiotic resistance genes at pulmonary exacerbation; ratio_FEV1, the ratio of the percent predicted forced expiratory volume in one second (ppFEV1) at the end of antibiotic treatment compared to the best ppFEV1 in the 6 months prior to the pulmonary exacerbation. In the multiple versions of the model ran, species observed was replaced by both Shannon diversity and the inverse Simpson index, while ratio_FEV1 was replaced by the improvement of ppFEV1 at end of treatment compared to the ppFEV1 at pulmonary exacerbation.